We recently identified and characterized small KCa (mSKCa) channels in the inner mitochondrial membrane (IMM) of guinea pig and human cardiac myocytes. A related class channel is the big mBKCa channel found in cardiac myocytes and other cell types. Reactive O2 species (ROS) are believed to be responsible for mediating protective effects of mKCa channel openers, but it is not known how mKCa channel opening acts to stimulate mild ROS generation or how this leads to reduced ROS production and better function during cardiac ischemia- reperfusion (IR) injury. Of the several known mKCa channels, all appear to be sensitive to one of the markers of ischemia (low ATP, low pH, low membrane potential, or high mCa2+). Because the function of each is to facilitate K+ entry into the matrix, the K+ uptake, and increase in mitochondrial volume, modulates mitochondrial bioenergetics in an unclear manner. Indeed, our published and preliminary studies show that activation of either the SKCa or the BKCa channel increases K+ uptake into the matrix, increases mitochondrial volume, and increased channel conductance in artificial lipid bilayers. Moreover, we found that mK+ influx induced by NS1619 (a BKCa activator) was accompanied first by an increase in matrix pH and then by a later decrease in matrix pH due to activation of K+/H+ exchange (KHE) as assessed by the KHE inhibitor quinine, which resulted only in a higher pH after application of NS1619. Also, BKCa channel opening with NS1619 led to increased respiration and H2O2 generation, but only with complex II substrate and blocked complex I electron flux. It is unclear how the individual mK+ channels are triggered by their respective ligands to promote K+ influx with IR injury (sensor), when these channels are opened during IR injury to initiate cardioprotective mechanisms (timing), why there are at least two conductance mKCa channels, and if the respective conductances are altered in mitochondria depending on the stimulation by the sensor (function). In particular, because we have identified a SKCa channel that is located in the mitochondrion, we are particularly well suited to compare the function and importance of this channel during cardiac IR injury. Our aims are to: 1: Determine isoform(s) that form mSKCa channels and assess their functional attributes (Ca2+ sensor, mitochondrial targeting sequence, conductance characteristics) when over/-under expressed in cell models or cloned &incorporated into lipid bilayers. This knowledge will lead to splice variant mitochondrial targeted drugs. 2: Assess cardiac function and regional infarct size in vivo in SKCa (SK2 and SK3) conditional mutant mice and mitochondrial and cell function in mutant cell lines and mutant mice. Assess cardiac function and bioener- getics in guinea pig hearts and isolated mitochondria SKCa vs. BKCa channel agonists or antagonists perfused before or after global ischemia, or only on reperfusion. 3: Simulate conditions of cardiac ischemia in isolated mitochondria and mitoplasts in human ventricular tis- sue in the presence or absence of KCa channel openers or antagonists.